Cytogenetic Studies of B-cell Chronic Lymphocytic Leukemia (B-CLL)

先前的g带核型分析发现，在适当的体外刺激多克隆b细胞有丝分裂原后，40- 100%的B-CLL患者中存在克隆性染色体异常。目前尚不清楚不同系列中报告的异常频率的广泛差异是由于疾病特征的真正差异还是仅仅是体外培养条件的差异。常见的复发性染色体异常包括12三体、14q32重排、13q易位或缺失、6q缺失和11q缺失。最近的研究表明，间期荧光原位杂交（FISH）技术增加了12、13q和11q三体缺失的检测，表明这些变化确实发生在疾病的早期。关于B-CLL核型的临床和预后意义的数据是有限的，在某些情况下是相互矛盾的。然而，初步临床研究表明，某些核型异常与B-CLL的特定临床病理亚群和病程有关。为了解决B-CLL核型变化的时间问题，以及这些变化可能的特异性和意义，我们正在对散发性或家族性B-CLL患者进行前瞻性研究，以进行评估和可能的治疗。本项目细胞遗传学的具体目的是：确定从B-CLL患者外周血中获得核型异常有丝分裂细胞的最佳培养条件；确定间期FISH是否检测到g带中期分析遗漏的克隆异常细胞；确定比较基因组杂交（CGH）是否能检测到中期或FISH分析未发现的染色体物质的增加或减少；并将细胞遗传学结果与疾病的临床、形态学和免疫表型特征、基因表达的cDNA微阵列分析以及新疗法的结果联系起来。从120例进入研究的患者中获得的数据显示，使用g带中期分析，只有30%的病例出现克隆性染色体异常，并证明大肠杆菌脂多糖作为白血病细胞的有丝分裂原的劣性。由5个探针组成的间期FISH证实或扩展了异常克隆患者的g带发现，并在迄今为止除10名“正常”或无g带信息的患者外的所有患者中检测到异常。从2002年到2005年，我们对先前进入研究的患者分离的DNA进行了回顾性的CGH，并对新患者的DNA进行了前瞻性的CGH。CGH检测亚显微缺失的灵敏度低于间期FISH，但检测到FISH未探测到的区域的增益和损失。结合g带、FISH和CGH，我们能够在超过90%的B-CLL患者中发现分子细胞遗传学异常。此外，在多个患者中使用两个探针进行双杂交和序列分析显示诊断时存在某些异常，并且在转化时存在额外的异常。现在，细胞遗传学结果与临床和其他实验室诊断和转化的特征，以及基因表达的cDNA微阵列分析和治疗结果相关。该项目正在进行中，它有可能产生关于临床细胞遗传学诊断技术的新信息，展示B-CLL患者的新临床病理亚群，并定义对B-CLL发生或进展至关重要的基因重排。

Previous G-banded karyotype analyses have detected clonal chromosome abnormalities in 40-100 percent of B-CLL patients studied after appropriate in-vitro stimulation with polyclonal B-cell mitogens. It is currently unknown whether the wide variations in frequencies of abnormalities reported in different series are due to true differences in disease characteristics or merely to in-vitro culture conditions. Common recurring chromosomal abnormalities include trisomy 12, rearrangements of 14q32, translocations or deletions of 13q, deletions of 6q, and deletions of 11q. Recent studies have shown increased detection of trisomy 12, 13q deletions and 11q deletions with interphase fluorescence in-situ hybridization (FISH) techniques, suggesting these changes do indeed occur early in the course of the disease. Data regarding the clinical and prognostic significance of karyotype in B-CLL are limited, and in some instances are conflicting. Nevertheless, preliminary clinical studies suggest certain karyotypic abnormalities are associated with specific clinicopathologic subsets of B-CLL, and with the course of the disease. To address the questions of timing of karyotypic changes in B-CLL, and the possible specificity and significance of these changes, we are conducting prospective studies of patients with sporadic or familial B-CLL referred to the NCI for evaluation and possible treatment. The cytogenetics specific aims of this project are: to determine the optimal culture conditions for obtaining karyotypically abnormal mitotic cells from peripheral blood of patients with B-CLL; to determine whether or not interphase FISH detects clonally abnormal cells missed by G-banded metaphase analysis; to determine whether or not comparative genomic hybridization (CGH) will detect gains or losses of chromosomal material not found by metaphase or FISH analyses; and to correlate cytogenetics results with clinical, morphologic and immunophenotypic features of the disease, with cDNA microarray analysis of gene expression, and with outcome with new therapies. Data from >120 patients entered on-study reveal clonal chromosome abnormalities in only 30 percent of cases using G-banded metaphase analysis, and demonstrate inferiority of e. coli lipopolysaccharide as a mitogen for the leukemic cells. Interphase FISH with a panel of five probes has confirmed or expanded the G-band findings in patients with abnormal clones, and has detected abnormalities in all but 10 patients tested to date who were "normal" or uninformative by G-banding. From 2002 through 2005 we performed CGH retrospectively on DNA isolated from patients previously entered on-study, and prospectively on DNA from new patients. CGH was less sensitive than interphase FISH in detecting submicroscopic deletions, but detected gains and losses of regions not probed by FISH. Combining G-banding, FISH, and CGH, we are able to find molecular cytogenetic abnormalities in more than 90% of B-CLL patients. Furthermore, dual hybridization with two probes and also sequential analyses in multiple patients have shown certain abnormalities present at diagnosis, and additional abnormalities at the time of transformation. The cytogenetics results are now being correlated with clinical and other laboratory features at diagnosis and transformation, and with cDNA microarray analysis of gene expression and outcome with treatment. This project, which has the potential to yield new information regarding clinical cytogenetics diagnostic techniques, to demonstrate new clinicopathologic subsets of B-CLL patients, and to define genetic rearrangements critical to initiation or progression of B-CLL, is ongoing.